1. Field of the Invention
The present invention relates in general to sample mounts for mounting and manipulating macromolecular and virus crystals and other samples for X-ray crystallography, and methods of using the same.
2. Description of the Background Art
One of the most common ways of mounting crystals for X-ray data collection and structure determination is to insert them into thin-walled (typically 10 micrometer) glass or quartz capillaries. These thin capillaries are X-ray transparent and produce relatively little background scatter. They can be sealed at both ends, providing a stable environment for the crystal. This is particularly important for crystals of proteins and other biological macromolecules, which contain large amounts of solvent (mostly water) and must be maintained in a constant humidity environment to preserve their structure and order. The environment of the crystal can be changed inside the capillary. For example, crystals can be controllably dehydrated by injecting a small amount of a saturated salt solution into the capillary. Crystals can also be soaked inside the capillary in solutions containing drug molecules, small molecule ligands, and heavy atom compounds. Capillary mounted crystals can be used for data collection from the melting point of the crystal solvent to well above room temperature. They are particularly important for crystals that cannot be flash frozen for data collection without inducing excessive crystal disorder.
In a known technique used to mount protein and other biomolecular crystals in capillaries, a capillary of diameter comparable to the crystal diameter is first selected, in order that the final mounted crystal lie near the center of the capillary to simplify alignment in the X-ray beam. Next, the sealed end of the capillary is scored and broken, and some means for producing suction attached to the other, larger diameter end. The open end of the capillary is inserted into the liquid drop in which the crystal resides and a small amount of this liquid is pulled into the capillary. The capillary is removed from the liquid and a small amount of air is pulled in to move the liquid away from the end. Next, the capillary is inserted back into the liquid drop and the crystal is carefully sucked in. Excess liquid surrounding the crystal is removed using paper wicks, and the open end of the capillary sealed with wax, grease, etc. The suction device is removed from the large diameter end, the capillary is scribed and broken to the desired length, the end is sealed and then the capillary is mounted onto a pin or goniometer head, typically using modeling clay, for X-ray measurements.
Another area where crystal mounting and manipulation is employed is in X-ray cryocrystallography, which is extensively used for crystals of proteins, protein-nucleic acid complexes and viruses and other crystals that are sensitive to radiation damage by X-rays. The development and application of cryocrystallographic techniques has had a dramatic impact on the rate at which structures of biological macromolecules and macromolecular complexes can be solved. Much larger X-ray doses can be absorbed before radiation damage becomes significant, so that complete data sets can often be collected using a single crystal.
A variety of methods have been used to manipulate mount crystals for flash cooling and cryocrystallographic data collection. Early experiments attached crystals to the ends of glass fibers or placed them on top of miniature glass spatulas. A loop mounting method using low X-ray absorption materials for the loop is now by far the most widely used method for manipulating and mounting crystals. Loop cryomounts consist of a small (10-20 μm) diameter nylon (or metal) line that is twisted to form a loop and then threaded into a small hollow metal rod. This rod is then inserted into a metal or plastic goniometer-compatible base. Crystals are retrieved from the mother liquor in which they are grown by capturing them in the loop, and then they are transferred using the loop between one or more solutions including stabilizing solutions, heavy atom compound solutions, solutions containing small molecules, drugs or ligands, or cryoprotectant solutions. Crystals larger than the loop can rest on its surface, while smaller crystals can be trapped in the liquid film that spans the loop or else adhered to the side of the loop. Loop-mounted crystals are then flash cooled by immersion in liquid nitrogen or propane or by insertion in a cold gas stream.
Loops provide convenient crystal manipulation. By holding crystals in the liquid film of the loop, potentially damaging contact with hard surfaces (such as those of alternative mounting tools) is minimized. The loop itself is flexible enough to make damage due to incidental contact less severe. Loops help minimize thermal mass and maximize surface area for heat transfer, increasing cooling rates and thus reducing cryoprotectant concentrations needed to prevent hexagonal ice formation within and surrounding the crystal. For these reasons loop-based mounts have been chosen as the standard for high-throughput automated cryocrystallography at synchrotron X-ray beam lines around the world.
The foregoing known techniques for mounting samples in crystallography applications have a number of drawbacks. In particular, the capillary mounting technique requires that both the initially sealed and wide diameter open end of the capillary must be cut and then subsequently sealed. These manipulations increase the chance that the capillary will be broken and the crystal inside damaged. In addition, the minimum capillary wall thickness that can be used and still provide adequate robustness for cutting is limited to about 10 micrometers. This in turn fixes the capillary's contribution to background X-ray scatter, which can significantly degrade the overall signal-to-noise ratio when measuring very small crystals.
For very small crystals (<50 micrometers), wicking away excess liquid without disturbing the crystal is difficult. Residual liquid between the crystal and capillary wall may have a volume comparable to the crystal volume (because of the larger surface to volume ratio of small crystals), which will increase background scattering of X-rays. Any residual liquid between the crystal and capillary also acts with the curved capillary wall as a distorting lens that makes accurate crystal alignment in the X-ray beam difficult. While some liquid is required to hold the crystal in place against the capillary wall, if there is excess liquid, the crystal may slip relative to the wall during diffraction measurements, which can create errors in data analysis.
To ensure that the crystal ends up near the radial center of the capillary, the capillary diameter must be matched to the crystal size. Consequently, capillaries of many diameters must be stocked. Even with a correctly sized capillary, the vertical and horizontal position of the crystal relative to the axis of the X-ray system is poorly controlled, requiring time-consuming alignment for each crystal. Positioning the crystal at a particular distance, e.g., from the base to be mounted in the goniometer head requires careful crystal manipulation and careful cutting of capillaries, which can be very time consuming. Adjustment of pressure in the suction device during crystal retrieval also requires considerable skill. Because of their large size, capillaries can obstruct the view of the crystal to be mounted, and are difficult to manipulate in small drops. Contact with the capillary ends and walls during retrieval often damages crystals (especially those with plate-like geometries), increasing their mosaicity and degrading their X-ray diffraction properties. Although possible, retrieving crystals from capillaries for further treatment or measurement is extremely difficult. Soaking crystals in ligands, drugs or heavy-atom compounds after data collection of a native structure can be performed in the capillary, but is inconvenient and often displaces the crystal.
In cryocrystallography, the use of loop mounts is also problematic. In particular, loops are quite flexible, especially those made using 10 μm diameter nylon line. As a result, loops can bend under liquid and surface tension forces during crystal retrieval from solution, and they can bend under the weight of the crystal and surrounding liquid once a crystal is mounted. Because of their irregular aerodynamic profile they can bend and flutter under the drag forces of the cryostream, slightly broadening X-ray diffraction peaks for the lowest mosaicity crystals and reducing the maximum diffraction signal-to-noise achieved when crystal mosaicity and incident beam divergence are matched.
In addition, loops provide poor crystal positioning accuracy and reproducibility relative to the X-ray spindle axis. The loop shape for a given nominal loop diameter is irregular and irreproducible. The loop orientation relative to the metal post through which they are threaded is irregular, in part due to the twist of the nylon at their base needed to improve rigidity. Crystal positioning within the loop is irreproducible, especially for very small crystals. The crystal and the loop itself (which gains rigidity from frozen liquid) can shift during in situ crystal “annealing” or “tempering” protocols that raise the sample temperature near or above the melting point/glass transition of the surrounding solvent, necessitating crystal realignment in the X-ray beam.
Loops can also trap significant liquid around the crystal. This liquid can be difficult to wick away, especially if the crystal is smaller than the loop's inner area. Remaining liquid increases background scattering, reducing diffraction signal-to-noise, and increases thermal mass, thereby decreasing cooling rates. Moreover, surrounding liquid has different freezing properties and thermal expansion behavior than the crystal and can exert damaging forces during cooling. Frozen surrounding liquid also can make small (less than 50 μm) crystals difficult to image and align.
The limitations of loops are becoming increasingly apparent as crystallographers attempt structural studies using smaller and smaller crystals made possible by continuing improvements in X-ray sources, optics and detectors. Initial crystallization trials—especially those based on high-throughput robotic screening—usually yield very small crystals. Collecting X-ray data from these crystals can provide valuable feedback early in the growth optimization process, and sometimes immediately yields useful structural information. Crystal size may remain small even after substantial optimization of crystal quality, especially in the case of macromolecular complexes and membrane proteins. Despite reduced signal-to-noise and increased radiation damage, smaller crystals may even be desirable because they flash cool more rapidly and thus are easier to cryoprotect; they can yield better diffraction data sets than larger crystals unless cryoprotection conditions for the latter are carefully optimized.
For crystals with sizes below 50 μm, loops become extremely difficult to use. Flexibility constraints limit the minimum nylon diameter, which in turn limits the minimum inner loop diameter. Smaller crystals must often be held in a liquid meniscus of larger volume, reducing diffraction signal-to-noise and making alignment more difficult. Large liquid-to-crystal volume ratios also limit reductions in thermal mass and cooling times.
In view of the foregoing, a need remains for improved techniques for mounting microcrystals for use in X-ray crystallography and cryocrystallography applications.